EP2

Question 1

Permeability to water when there is no osmotic gradient?

Answer 1

Diffusional water permeability (Pd).
Continuous movement into/out of the cell.
Influx=efflux. No volume change.

Question 2

Permeability to water when there is an osmotic gradient?

Answer 2

Osmotic water permeability (Pf) e.g. cell in hypertonic solution.

Question 3

Pf/Pd ratio?

Answer 3

Directional water movement/ bidirectional water movement.
If ratio greater than one: There is a water pore present in cell membrane (as directional movement).
e.g. NA/glu cotransporter has water pore properties.

Question 4

Study on Pd/pf in cells? Vague method and results?

Answer 4

1953 mesured Pd/pf in a variety of cells using the cartesian diver balance method.
Greater than 1 (water pore) in all cells tested bar the trout eggs.

Question 5

How to measure Pd?

Answer 5

Cartesian Diver Balance.
Diver balances at a varaible equilibirum in a heavy water (D2O) chamber according to cell weight and pressure of chamber.
As cells exchange D2O for H2O they will pull the diver down in the chamber as they get heavier. The amount of pressure needed to be added into the chamber to keep the diver at a given height is measured. (Add vaccum diver rises).
suction needed proportional to the water diffusion.

Question 6

1953 cell study Pd results?

Answer 6

All cells (bar trout) had to keep having suction put into the chamber but less and less over time as the diver gradually equilibriates where the same amount of D2O and H2O is transferred. On log graph there is a gradual plateau. 
Rapid exchange of cell water in all cases with the t½ of exchange < 4.5 minutes bar In trout eggs no evidence of water diffusion even after 5 hours

Question 7

How to measure Pf?

Answer 7

Meaure the change in volume of a cell overtime when exposed to hypertonic/hypotonic solutions.

Pf= AV/ SA x t x AC
Change in vol (diameter)= Pf x Surface area x time x concentration change (gradient)

Question 8

1953 cell study Pf?

Answer 8

All cells swelled when added hypotonic solution in the bath (low solutes outside- water rushes in the cell) and shrinks when hypertonic (increased solutes outside- water leaves).
All cells bar the trout egg.

Question 9

Explaination for trout eggs very low (no) water permeability ability?

Answer 9

They are layed into freshwater (not saltwater), so likely evolved to lose this ability else water will just rush in and the egg will swell and burst.

Question 10

Red blood cell water permeability is very..? Therefore..

Answer 10

High.
Pf/Pd ration =2.5.
This predicts a water pore in. (was very hard to isolate)

Question 11

How was the RBC water pore eventually found? (part 1- until AB to)

Answer 11

Agre group found 1991:
Was studying the Rheus protein in RBCs but kept getting a 28kD proein co-precipitating. (1988)
Isolating this protein was hard but producted an antibody to the 28kD protein.
This AB also recognised a higher MW band (but not the 32KD rheus protein) and 2/3 times the size (exists as tetromers- cryoEM confirmed).
If treated with a deglycosylation enzyme- drop back to 28kD.
AB stained PT and thin decending limb (high water permeability)

Question 12

How was the RBC water pore eventually found? (part 2- until calcualtions)

Answer 12

Agre group 1991:
The N terminus of the 28kD protein was sequenced (from human faetal liver) so PCR and library screening could be used to identify CHIP28 (AQP1).
Sequence analysis predicted 6TMD and 42% homology to waterchannel in the eye (AQP0- makes up 60% of lens is).
Both had tandem NPA repeats (suggested critical form
water pore formation).
Biophysical Calculations of water channels needed and number of CHIP28 in RBCs added up. And both resistant to enymatic digestion.

Question 13

How was CHIP28 (AQP1) proven to be a water chanel?

Answer 13

1992:
CHIP-28 was expressed in xenopus oocytes and then the oocytes exposed to a hypertonic shock
In control oocytes the volume change is very slow.
In oocytes expressing CHIP-28 the volume change is rapid and the oocytes explode with in a few minutes. Renamed AQP1.

Question 14

AQP1 is inhibited by? Ecidence?

Answer 14

Mercury sensitive.
1993 study.
Measured the volume of xenopus oocytes expressing AQP1 when exposed to hypertonic shock (water inflow) . Volume increase of 35%.
If add Hg- volume increase decreased to 15%.
If add ME (beta Mercatoethanol reducing agent which binds to cysteins so mercury cant) back to 35%.
Xenopus without AQP1 expression vol increase of 5%.

Question 15

How does mercury inhbit AQP1? Evidence (2)? (not specific number)

Answer 15

Mercury binds to exposed cysteine residues of AQP1.
Know because treatment of B Mercatoethanol binds to cysteins so HgCl2 cant stopping the Mg inhibition (volume returns to 35% increase after 15% with Mg).

Also, If mutate 4 cysteins in the AQP1 and replace any with serine the water permeability increases after Hg treatment compared to when cysteins (cant bind and inhibit) But no impact on water permeability without Hg.

Question 16

WHich aquaporin is mercury insensitive?

Answer 16

AQP4.

Question 17

Exposed cystein that Mercury binds to? Evidence? (x2)

Answer 17

Cystein 189.
1993 Preston: If mutated cystein residues individually and see impact on mercury on the water permeability.
Results:
Normal function of AQP1 without HgCl2.
HgCl2 still reduced AQP1 function with mutated 3 of the cysteins, but not C189.

Savage 2007 study: Also.. If take bacterial AQ1 (HgCl2 insensitive) and engineer a C189 to it- forms crystals with Hg and now mercury sensitive.

Question 18

AQP1 stucture?

Answer 18

6TMDs, with the B and E (between 2-3 and 5-6) loops not crossing the membrane but dipping into. These have the NPA motifs.
These two dipping loops cross over each other and make the pore between. This creates an hourglass like model.
4 subunits come together to give the tetromer- each making a pore through.

Question 19

AQP1 have 4 pores or one central? Experiment method?

Answer 19

Made of 4 subunits coming together (6TMDs and have a pore in each).
Jung 1994. Made tandem dimers of either AQP1 subunits or C189s mutants.
mRNA injected therefore will be mix, could be all WT, all mutant or mix etc.
If mix would it give an intermediate phenotype ie 3/4 mutant- have 3/4 pores working etc, or if central pore will be all or nothing function.

Question 20

AQP1 have 4 pores or one central? Experiment results?

Answer 20

Yung 1994-
Without mercury all channels gave functional water channels.
WT- mercury brings back to baseline levels.
Gave intermediate (25%, 50%, 75%) mercury sensitivities when mixed WT and mutant C189S subunits.
Concluded: Must be individual functional channels

Question 21

WHat is surprising about how water moves through aquaporins?

Answer 21

There is no piggybacking of protons through, which would be expected, however there is a single file movement of water through the pore.

Question 22

How is water transported in single file through aquaporins? Model for?

Answer 22

Predicted that watermolecules H+ bonds are broken and they bind to the NPA motifs through new H+ bonds which ensures selective water movement through which inhibits protons piggybacking.

Question 23

Experiment of single file water movement through AQP1? Method:

Answer 23

NSF motifs in B (Asn-Pro–Ala) and E and a hydrophobic lining of the pathway for the water through.
Stochastic boundary molecular-dynamics (SBMD) method was used to probe the dynamics of particles ad the H2O moved through.

Question 24

Experiment of single file water movement through AQP1? Results:

Answer 24

It was seen that the distribution of water density near the NPA motifs is significantly thinner, confirming that the narrower space around that region imposes a constriction to the flow of water.
Also hydrogen bonds with the polar (hydrophillic) side chains of NPA could be seen on the stereochemistry diagrams and were found to be essential. When in B loop the Asn and Asn were replaced with near-identical hydrophobic residues in the simulation, the aqueous pathways were broken completely.

Question 25

Polar is hydrophobic or phillic?

Answer 25

Hydrophillic. (polar bears love swimming in water to get fish)

Question 26

Experiment of single file water movement through AQP1? Discussion:

Answer 26

``` Discussion: The size restriction on molecules to pass by the narrowing through the NPA will limit what goes through the channel and promote the single file pasage. Importantly, the size of the constriction fluctuates significantly, which frequently breaks the flow of water, thus breaking the single-file water network necessary for proton translocation The polar(hydrophillic) NFA residues are essential to H bind to the water molecules and ensure H+ ions cannot pass alone. ```

Question 27

AQP3 channel for? Found?

Answer 27

If express in xenopus oocytes, increase the mercury sensitive passage of water through. Also a Hg++ sensitive increase in glycerol and urea permeability. Could measure when radiolabelled. Found: In basolateral collecting duct to reabsorb water for example.

Question 28

AQP6 channel for? Found?

Answer 28

Unorthodox aquaporin. Found in the kidney and colocalises with H+ ATPase proton pump in in alpha intercalated cells. MERCURY STIMULATED. water permeability increases with. Cl- conductance also. PH SENSITIVE also- stimulated at low PH ( if H+ in (acidic) Cl- will also come in to balance charge)

Question 29

AQP6 stimulated by?

Answer 29

``` Mercury. Low PH (acidic- Cl- in to balance charge) ```

Question 30

Classes of MIP proteins?

Answer 30

1) Aquaporins e.g. AQP 0,1,2,4,5 (water only) 2) Aquaglyceroporins e.g. 3, 7,9, 10 (+glycerol and urea) 3) Unorthodox e.g. 6, 8, 11, 12 (NPA changes to NPS) 13 in humans.

Question 31

AQP8 channel for?

Answer 31

linked to ammonia transport

Question 32

AQP2 mutation?

Answer 32

Diabetes insipidis. Cannot concentrate their urine- lose 20l urine a day. Vasopressin doesn't help (inserts more AQP2 into the apical membrane of the collecting duct. These mutations may affect trafficking or permeability to H20. AQP3 and 4 on basolateral.

Question 33

Aquaporins in epithelia?

Answer 33

Reabsorptive Epithelia – Proximal tubule – AQP1 Secretive Epithelia – Salivary Gland – AQP5 Reabsorptive Epithelia – Small Intestine – No AQPs

Question 34

Importance of AQP1? Study

Answer 34

Hand dissected out kindey and mount on micropipettes. and measure water permeability. KO AQP1 in proximal tubule: water permeability decreased by 78%. (Pf) water cant move across to balance the hypotonic gradient in lumen created, therefore the gradient is much larger (from around 10mOsmol/L to around 40mOsmol/L)

Question 35

How does water reabsorption in proximal tubule occur?

Answer 35

At near iso-osmotic conditions. Na and Cl out of the lumen into the epithelial cells, leaving the lume slightly hypotonic. Water follows and the epithelial cells reabsorb water in through AQP1 (basolateral). But extremlely high water permeability of the tubule means this is sufficient.

Question 36

PCT cell what channels are on Apical and basolateral?

Answer 36

Apical: Na/H exchanger and H recycled back in. CO2 diffuses in. Basolateral: Na/HCO3 cotransporter and AQP1.

Question 37

How does microperfusion experiments occur in PCT?

Answer 37

Dissect out PCT with the outer of epithelial cells and inner lumen. Liquid in lumen= perfusate (what is added) and liquid outside the epithelial cells=absorbate (what is reabsorbed from PCT). Oil droplet is put around the vessel on basolateral side of ECs and this makes up the absorbate. As the cells reabsorb, a water droplet forms under the oil droplet= absorbate. Can remove this oil and measure its osmolality difference between the two. Meausre H2O gradient. around

Question 38

Experiments to prove PCT water reabsorption near-isotonic?

Answer 38

16mosmol/kg (near iso-osmotic) PCT H2O gradient difference from perfusate to absorbate using microperfusion. Micropuncture experiment-

Question 39

Micropuncture technique?

Answer 39

In vivo. Pipettes put into capillaries of anaethasised animals. Known NaCL (no HCO which also drives water reabsorption though) solution concentration into PCT lumen and capillaries and the PCT fluid was collected.

Question 40

micropuncture technique results?

Answer 40

Less than 2mM gradient drives lots of water reabsorption. Low flow rate: slightly hypotonic PCT fluid collected compared to the capillary. High flow rate: rate of absorption higher.

Question 41

Why is transport in the PCT slower in high flow rates?

Answer 41

Fluid comes to equilibrium in the PCT, at slower flow rate reaches equilibrium faster (gradient neutralised), so no more fluid can be absorbed (e.g. Gordon cooper walkign across lecture us with paintguns, shoot him all over if going slowly, less if fast.) Whereas in slower flow rates, gradient persists for longer and so more water reabsorbtion can occur.

Question 42

Secretive epithelia example?

Answer 42

Acinar cell. Salivary gland. Has AQP5.

Question 43

secretive epithelia how works?

Answer 43

Secretes Na and Cl- into lumen- creating a hypertonic lumen. This creates an osmotic gradient for water out of the cell through AQP5 (balance out). Uptake on basolateral through NKCC1 (Na and Cl- into cell) and Na paracellularly.

Question 44

AQP5 KO?

Answer 44

Measured salivary secretion after Pilocarpine (stimulates salivary secretion) Measure vol of secretion and osmotic conditions. WT- high secretion, osmalarity near isotonic. (water balances) AQP5 KO- less than half and high osmolality- highly concentrated saliva (low water content)

Question 45

Whats odd about water reabsorption in the small intestine?

Answer 45

Apical side lacks any aquaporins and is quite tight. Has a high osmolality (500-550 mOsm) in lumen as releasing Na, Cl, AA, proteins after food broken down. This would drive water secretion. But uphill reabsorbtion against gradient (200mOsm gradient). SOMETHING MUST BE ACTIVE. Some transporters also transport water.

Question 46

Hyper and hypotonic?

Answer 46

Hypertonic- high solute concentration, high osmolality- water will move into. Hypotonic- low solute concentration, low osmolality, water will move out of.

Question 47

Epithelia that are reabsorbing water? Secreting?

Answer 47

Reabsorbing: Kidney PCT CD, small intestine Secretory: airway

Question 48

Cotransporters that also transport water?

Answer 48

KCC4- 500molecules of H2O every pass NKCC1- 590 (basolateral of cells e.g. salivary) GLUT2- 40-100

Question 49

NKCC1 or 2?

Answer 49

1- basolateral of cells e.g. salivary glands (has water transport role) 2- kidney e.g. thick ascending limb

Question 50

Study evidence for water transport with other cotransporters?

Answer 50

Frog cell expressing KCC transporter, measure cell shrinkage/growth after hypotonic shock (graidnt increase 100mOsm) with mannitol 100mM). generally cells shrink as water moves out. Add NaClmM (100mOsmol change) shrink again. Add 50mM of KCl. Now cells swell. Because KCl wants to move into the cell against gradient and water comes in with the KCC4 channel even against the osmolality gradient. And Furosamide (Inhibit KCC and NKCC proteins), cell shrinks again, so is through this channel.

Question 51

Water moves throuh aquaporins through.? Through other channels by?

Answer 51

Aquaporins: Diffusion. Channels: cotransportation Mix of both, SGLT EAAT1 (amino acid transporter)

Question 52

Which channels have a mix of diffusion and cotransportation through channels? How?

Answer 52

SGLT (Na glucose cotransporter) EAAT1- amino acid transporter Creates microgradients where transport solutes across in very thin layers next to transporters, creating gradients of water. So say sodium moves into cleft, creates gradient and water will move in wth glucose- conformational change and moved across.

Question 53

Water reabsorption in the small intestine channels?

Answer 53

APICAL: 500mOsm. SGLT2 brings in Na, glucose, H2O and GLUT2 brings in Glucose and H2O from the lumen. Basolateral: 300mOsm. Na/K ATPase(Na out, K in). KCC (K recycles, Cl- into the blood as well as H2O), GLUT2 also brings glucose and H20 in reabsorbing.

Question 54

AQUAPORIN1 also has a channel for..? relevence?

Answer 54

Gas. | Red blood cells and colton null people important in

Question 55

What was thought before Overtons law? Why?

Answer 55

It was previously thought that membranes were fully permeable to solutes/gases. Because O2 or CO2 have a high solubility in oil so are freely permeable so it was presumed that all membranes were freely permeable to all gases. Also, artificial membranes were freely permeable but these had no cholesterol or membrane proteins and decane preparation solvent increased fluidity.

Question 56

What is overtons law?

Answer 56

Overton realised the permeability of membranes to gases/solutes was proportional to the oil/water partition co-efficient for that solute.

Question 57

Experiment to test overtons law?

Answer 57

See how well different solutes dissolved in oil vs water to test the theory that permeability of membranes to gases/solutes was proportional to the oil/water partition co-efficient for that solute.

Question 58

NH3 causes cells PH to? | NH4 causes cells PH to?

Answer 58

NH3(ammonia)- PH rises (alkaline) as NH3 combines with free H+ ions in the cell to become NH4. NH4(ammonium)-PH falls (acidification) as NH4 dissociates into NH3 + H+. But slower uptake of ammonium into cells, as isnt permeable to.

Question 59

Are membranes less permeable to ammonia or ammonium?

Answer 59

Ammonium (NH4), Isnt permeable to across bilayers, but is transported through transporters like NKCC (in thick ascending limb) instead of K.

Question 60

CO2 or HCO3 into a cell impacts PH how?

Answer 60

Co2 moves into cells, combines with H2O= HCO3+H+ (acidification) HCO3 moves into cells, combines with H+ to make CO2 and H20- uses up free H+, alkalisation.

Question 61

If cell there is acidification transported in is? (x2) | Alkalisation? (x2)

Answer 61

Acidification: Ammonium NH4 or CO2.- (generates H+) Alkalisation: Ammonia (NH3) or HCO3

Question 62

If cell there is acidification transported in is? (x2) | Alkalisation? (x2)

Answer 62

Acidification: Ammonium NH4 or CO2 (generates H+) Alkalisation: Ammonia (NH3 gas) or HCO3-

Question 63

First experiment that evidenced low gas permeability?

Answer 63

Permeability to NH4/NH3 in thick ascending limb. Measured the PH of cells, and if Ammonium chloride is added to the basolateral side see an alkalisation, predictable permeability to ammonia and ammonium. but to the apical see an acidification, high permeability to NH4 but very low NH3 (gas).

Question 64

Follow up experiment that evidenced low gas permeability?

Answer 64

Gastric gland permeability to CO2/HCo3. Dissected out gastric glands and put on micropipettes if Co2 put on basolateral side- acidification (as expected) but if 100% Co2 added on lumen side- no PH change- impermeable to gas. Same result in colon crypt.

Question 65

Why may the gas impermiability make sense in gastric glands?

Answer 65

Stomach PH very low HCL- near to 1 very acidic. So having the apical membrane impermeable to gases is protecting stomach from digesting itself, else the cells would become hugely acidic and not be able to survive.

Question 66

What causes low gas permeabiltiy of membranes? Experiments (2)?

Answer 66

Linked to cholesterol content. 1. Experiments using liposomes artificial bilayer, can change the cholesterol content in. - At low cholesterol levels: High Co2 permeability freely - As increased into physiological range- linear decrease in Co2 permeability. 2. MDCK kidney cultured cells, measured CO2 permeability. If strip out cholesterol from membrane, CO2 permeability increased massibvely, and if increased- reduced.

Question 67

Cholesterol content level in thick ascending limb? Collecting duct?

Answer 67

TAL: predict v high cholesterol due to low gas permeability. CD: high cholesterol but aquaporins so tight but regulated passage.

Question 68

Cancer cells cholesterol content?

Answer 68

Low (30%), as they produce Co2 which needs to be lost and O2 to diffuse in to meet metabolic demands (very active cells).

Question 69

Colon crypt cholesterol content?

Answer 69

77% cholesterol in apical as well as proteins in there to reduce surface area for gas movement. Glycolipids and glycoproteins to reduce also.

Question 70

RBCs cholesterol content?

Answer 70

Apical: 45% but this wouldnt support the amount of Co2 transport that has been measured. Proteins and transporters also.

Question 71

Proximal tubule cholesterol content?

Answer 71

Apical: 45% but this wouldnt support the amount of Co2 transport that has been measured. Proteins and transporters also.

Question 72

WHy use the xenpus oocytes in experiments into Co2 permeability?

Answer 72

If add a Co2/HCO solution know its gas movement as there are no native bicarbonate transporters in the xenopus oocyte. = Gives acidification and this is proportional to the CO2 permeability (without bacground bicarbonate permeabilityy).

Question 73

How to measure CO2 permeability in Xenopus oocytes?

Answer 73

If add CO2/HCO solution (acidification) and then add water, can measure how long it takes the cell to swell and explode. Co2 permeability is proportional to water permeability. The more AQP1 channels there were (and faster lysis time), the faster the acidification was.

Question 74

what factor can you use to measure CO2 permeability in the xenopus oocyte (not water)

Answer 74

If add a Co2/HCO solution know its gas movement as there are no native bicarbonate transporters in the xenopus oocyte. = Gives acidification and this is proportional to the CO2 permeability (without bacground bicarbonate permeabilityy).

Question 75

How can one check the Co2 permeability experiments in xenopus oocytes are not just the aquaporins making the membrane more leaky?

Answer 75

If add an organic mercurial agent which inhibits the AQP1, CO2 permeability decreases. Also repeat this with C189s mutant, the CO2 permeability is returned with mercurial agent. First experiment proving that gas permeability is linked to protein channels.

Question 76

what are the group of people called with no AQP1?

Answer 76

Colton null blood group

Question 77

How much of CO2 permeability is due to AQP1? How do we know?

Answer 77

Colton null blood group donated some RBCs. These lack AQP1. CO2 permeability was measured and this was reduced by 50%. So roughly 50% of the CO2 permeabilty is due to AQP1. ( using mercurial agent had no impact also)

Question 78

As well as AQP1 how else is CO2 passing through RBC membrane? Experiment?

Answer 78

DIDs blocks CO2 permeability through AQP1 but not water permeability. In WT- reduced this by around 1/3. Colton null- reduced the Co2 permeabiltiy even further than already was (50% of WT already). DIDs inhibits rheus protein in RBCs- so around 40-50% due to this. 10% left through lipid bilayer

Question 79

What goes through each pore in AQP1?

Answer 79

``` x4 outerpores (in each subunit) Water and Co2(40%). Have both hydrophillic and hydrophobic domains in. x1 central pore- Co2 (60% ) and O2 (85% through central , 15% through outer). Mainly hydrophobic and gated by hydrophobic residues which prevent water access. ```

Question 80

AQP4 permeability to Co2?

Answer 80

M1- Kidney- has no permeability (in full length protein whereas in truncated M23 does) M23 in BBB- Co2 permeability- this will help with gas movement as this an extemely tight.

Question 81

Under what chemical can AQP1 not become CO2 permeable.

Answer 81

AQP1 binding of cGMP to, turns it into an anion channel throught the central pore. Oocytes treated with cGMP, AQP1 or 5 will lose Co2 permeability. cGMP changes conformation so CO2 cant go through but cations can- but no impact on water permeability. REGULATION.

Question 82

AQP5 where? function?

Answer 82

Also has Co2 movement through. Expressed in type 1 pneumocytes in the lungs to allow gaseous exhange into/out of the blood.

Question 83

Small intestine has no water porins yet it reabsorbs... liters of fluid daily. How?

Answer 83

Around 8 liters a day. | SGLT1 can cotransport water with the ions.

Question 84

Method to measure water through SGLT1?

Answer 84

We overexpressed SGLT1 (eGFP tagged) in MDCK cell monolayers and reconstituted the purified transporter into proteoliposomes. We observed the rate of osmotic proteoliposome deflation by light scattering i.e. if they deflate water has left the liposome. After measuring the SGLT1 abundance on MDCK cells and concentrations either side of the monolaters membranes, the unitary water channel permeability, pf was measured. This yielded similar values for cell and proteoliposome experiments so was deemed a suitable model.

Question 85

Aim of the SGLT1 water through experiment?

Answer 85

Previously suggested that high solute amounts passing through the SGLT1 channel would inhibit water through. But also wanted to test whether no water could still pass without Na or glucose. AIm to find out how they are all transported in the channel, together or separate?

Question 86

Result of the SGLT1 water through experiment?

Answer 86

Neither the absence of glucose or Na+, nor the lack of membrane voltage in vesicles, nor the directionality of water flow grossly altered pf. Not the limiting step for water movement through? Addition of Na had no impact on the Pf but 20 mm glucose addition resulted in a 17% drop in pf , suggesting that at least some of the water molecules share the pathway of glucose and are delayed by its presence.

Question 87

conclusion of the SGLT1 water through experiment

Answer 87

Such weak dependence on protein conformation indicates that a water-impermeable occluded state (glucose and Na+ in their binding pockets) lasts for only a minor fraction of the transport cycle or, alternatively, that occlusion of the substrate does not render the transporter water-impermeable as was previously suggested. Water partially shares the glucose route, and thechannel is permeable to water when empty, and with solutes (definitely Na). The high Pf levels of the SGLT1 excludes the need for active pumping in the small intestine inspite of against the gradient.

Question 88

Proximal tubule PH? Why?

Answer 88

PCT PH 7.4. | with Plasma HCo3 around 25mM and 5% Co2 (1.2mM dissolved).

Question 89

Most of the reabsorbed HCO3 from kidney is where?

Answer 89

80% in the PCT.

Question 90

APical movement of HCO3 PCT reabsorbtion is due to?

Answer 90

CO2 reabsorption across the PCT and H20, which cab combine to make HCO3 inside the cell.

Question 91

How is HCO3 reabsorbed over the PCT?

Answer 91

In lumen HCO3- (+H) to make carbonic acid, which then dissociates into H2O and CO2 under carbonic anhydrase. Co2 can move across the membrane (CO2 in AQP1). water already in. When inside the cell these combine to make HCO3- + H+ (carbonic anhydrase). The H+ recycles across the membrane by NHE3 exchanger (out) and active proton pump into cell. THe HCO3- then leaves over the basolateral side with Na (1Na: 3HCO3-)

Question 92

Where are the different carbonic anhydrases?

Answer 92

Type IV in lumen of PCT. Tethered to apical membrane. | Type II in PCT cells.

Question 93

NHE3 stoichiometry important why?

Answer 93

1Na: 3HCO3- NHE3 in PCT- need for reabsorption. | Whereas in pancreas 1Na: 2HCO- drives uptake of 2HCO3- into the cell

Question 94

What factors stimulate HCO3- reabsorbtion?

Answer 94

Angiotensin II Endothelin 2 NorA, Adenosin ALL INCREASE CA AND CAUSE PKC ACTIVATION

Question 95

What factors downregulate HCO3- reabsorption?

Answer 95

ANP Dopamine Perathormone INCREASE CAMP- PKA ACTIVATION

Question 96

If high levels of ... in blood, HCO3- reabsorption is..

Answer 96

CO2 high in blood, HCO3- reabsorption is inhibited | The level of CO2 in blood regulates HCO3- reabsorbtion across the PCT.

Question 97

Experiment to test whether CO2 is regulating HCO3 reabsorption across the PCT?

Answer 97

Out of equilibrium Co2/HCO3- solutions. Add pure Co2 and no HCO3- or pure HCO3- and no CO2. If add HCO3- going to get Co2 as dissociates etc, so wanted to design a solution which were at 100% at 7.4PH (out of equilibirum where normal PH 7.4 blood 5% CO2 and 22mM HCO3-)

Question 98

How were the out of equilibrium solutions made?

Answer 98

Solution 1: a. Solution at 10% Co2 at acidic PH (5.4), V low HCO b. Other syringe 100% oxygen, no Co2 or HCO- PH 7.55 but heavily buffered. MIX with mesh (turbulent flow)= Pure Co2 PH 7.4 normal 5% Co2 and no HCO for few hundred milisecond. ``` Solution 2: a. slightly acidic solution of no CO2 or HCO. b. 44mM HCO3 of alkaline PH MIX= Pure HCO3- 7.4 PH. (NaOH buffer ensures no CO2 dissolved) ```

Question 99

Pure CO2/HCO out of equilibirum results?

Answer 99

Used the pure CO2 or pure HCO3- solutions to run through PCTs to compare reabsorbtion solution on basolateral membrane. Removing HCO3- increased HCO3 reabsorption (when normal Co2). Increase driving force and preventing backflux through tight junctions. When no Co2- HCO3 reabsorption decreased. Basolateral blood CO2 sensor in PCT?

Question 100

CO2 sensor for HCO3- reabsorption works how?

Answer 100

Basolateral PCT Co2 sensor. If chnage concentrations in lumen- no impact but change in bath Ca increased in PCT cells. Co2 sensor on basolateral side (in blood). Repeated with pure HCO3- no change in Ca, but added pure Co2- got change in Ca, so looks like this is what is being sensed.

Question 101

Possible identity for the basolateral CO2 sensor in PCT?

Answer 101

Receptor protein tyrosine phosphatase Y (membrane linked).

Question 102

Evidence that it is Receptor protein tyrosine phosphatase Y that is the PCT basolateral CO2 sensor?

Answer 102

1. If this is inhibited by Receptor tyrosine kinase inhibits, the link between CO2 levels and HCO3- reabsorption was abolished. 2. Localisation experiments-present in the PCT at basolateral membrane. 3. -/- KO RPTP mouse- look at HCO3- PCT transport. Fixed PH and HCO3- but changed CO2 levels- inhibited HCO3- transport generally. And this linear relationship was lost. HCO3- changes no reponse either (WT does) 4. Phosphatase binding domain faces towards blood has homology to carbonic anhydrase site where CO2/HCO3- can bind to.

Question 103

Physiological reason for HCO3- reabsorption control by CO2 in blood?

Answer 103

High Co2 in blood- in acidic condition(acidosis). If reabsorb more HCO3- across PCT- balance Ph (HCO3 can sequest H+)

Question 104

Theory of how the CO2 HCO3- PCT sensor works?

Answer 104

CO2 and HCO3- binding to receptor protein tyrosine phosphatase Y- inhibition of its phosphatase activity- the Receptor protein kinases activity will dominate.

Question 105

Why is bicarbonate transport important?

Answer 105

Helps drive water reabsorption. | Buffers PH.

Question 106

Bicarbonate cycle of dissociation etc?

Answer 106

HCO3- + H+ = H2CO3 (carbonic acid). | H2CO3= CO2 +H2O dissociates under carbonic adhydrase enzyme.

Question 107

Method for measuring link between HCO2- reabsorption and water?

Answer 107

Rabbit PCT, injected an oil drop into the PCT lumen (stop perfusion either end of tube), and inject fluid in the middle (can change composition). Measure volume of this fliud drop in PCT, will shrink as cell reabsorbs water etc. (split drop micropuncture technique). Proportional to water flux Pf.

Question 108

Results for measuring link between HCO2- reabsorption and water?

Answer 108

If high HCO3- composition in lumen- high water reabsorption, whereas if remove the HCO3 the shrinkage of the waterdroplet in the lumen decreased.

Question 109

Role of the NHE3 in HCO3- reabsorption?

Answer 109

KO mouse for NHE3 (Na and HCO3- over basolateral membrane to reabsorb in PCT). Microperfusion technique saw a decrease in plasma PH (more acidic as HCO3- decreases so doesnt sequest H+ ions). HCO3 transport down by 60%. Water flux also decreased by around 70%.

Question 110

NHE3 in PCT drives ..... HCO3- reabsorption and ..... water.

Answer 110

60% | 70%

Question 111

HCO3- is ....in the pancreatic duct?

Answer 111

secreted

Question 112

Model of pancreatic duct for HCO3- secretion in rats?

Answer 112

On apical membrane- Cl- HCO3- exchanger secreting HCO3-. Cl- is recycled as it is pumped back out through CFTR. On the basolateral membrane- NHE (Na in). Co2 moves through, combines with H2O- HCO3- leaves across apical and H recycled across basolateral.

Question 113

Why is the pancreatic secretion model different for other mammals that arent rats?

Answer 113

MOre simple in rats as their lumenal HCO3- is only 70mM, but other mammals upto 140mM (humans ginea pigs etc)- electrochemical gradient to move back into cell, Cl/HCO3- shuts down.

Question 114

How is the HCO-3 secretion in non rat mammals achieved?

Answer 114

Against electrochemical gradient, Electrogenic Na/HCO3- cotransporter- Na 1: 2HCO3- into the cell. Brings into the cell over basolateral membrane, building concenration up in cell so favour secretion. Also HCO3- secreted out through CFTR.

Question 115

Evidence for apical secretion of HCO3- through CFTR in pancreatic duct? 2009 Method:

Answer 115

Isolated the pancreatic duct in WT and AF508 mice. Controlled luminal and baso contents and measured the PH inside cell. If HCO3- moves into cell= Alkalisation (binds to protons) If HCO3- moves out of cell= acidification. Set up solutions: Baso Cl- and HCO free and added DIDS (inhibit Na/HCO3-) and CAMP to stimulate CFTR. Lumen CL- free but high HCO3- concentration. Step changed K concentration (changes membrane potential)

Question 116

Evidence for apical secretion of HCO3- through CFTR in pancreatic duct? 2009 Results:

Answer 116

Increased K conc- depolarised the cell, HCO3- moved through CFTR (secretion) = Acidification. If hyperpolarise-HCO3 moving in= Alkalisation. Repeat in AF508- no PH changes at all-therefore Ph changes driven by HCO3- movement through CFTR.

Question 117

As go down the pancreatic duct how does secretion were?

Answer 117

Acinar cells secrete a Cl rich secretion. NKCC1 in basolateral causes Cl uptake, and Cl and Na secreted across the apical membrane (sectreted) As fluid moves down the duct Cl is reabsorbed by intercalating duct cells and replaced by HCO3- secretion. Early in duct SLC26 (anion exchanger including Cl- in, HCO3- out) and CFTR (HCO3 and Cl out) combination on apical membrane. But CFTR inhibits CL/HCO30 exchanger further down the duct and HCO3- is mainly secreted.

Question 118

CFTR and SLC26 interactions?

Answer 118

SLC26 interactions with CFTR. At rest- both inactive. CFTR NT binding domains inhibiting CFTR. With PKA stimulation, P of regulatory domain, CFTR inhibition is released so activated. Interaction with regulatory domain of SLC26, which promotes the Cl/HCO3- exchange (recycling of Cl- acorss membrane while secreting HCO3-).

Question 119

Why is SLC26 not simply working to secrete HCO3- in experiments?

Answer 119

EXperiments were done in CL- free conditions, and this needs Cl- to exchange- so its CFTR mainly. Also CF mutations affect HCO3- transport.

Question 120

Further evidence of CFTRs role in transporting HCO3- and that it is a different mechanism to Cl-?

Answer 120

CF patients with pancreatic insufficiency or sufficiency were studied. If stimulate WT CFTR with forskolin get Cl- movement= current. Also, PH change as HCO3- secreted. I148T mutation- normal Cl- transport but HCO3- disrupted- insufficient. R117H- slight Cl disruption but HCO3- retained= sufficient.

Question 121

NBC?

Answer 121

The electrogenic sodium bicarbonate cotransporter

Question 122

NBC splice variants?

Answer 122

kNBC: Kidney PCT- 1Na into blood: 3 HCO3 into blood -ABSORBTION HCO3- pNBC: Pancreatic- 1Na into cell: 2 HCO3 into lumen- SECRETION HCO3 (N terminus difference)

Question 123

Does the N terminus account for the stoichiometry difference of NBC? Method:

Answer 123

Compared a PCT cell line (reabsorb) and cortical collecting duct cell lines (secrete) with no NBC activity. And expressed pNBC and kNBC in both transfection. Mounted in using chambers and voltage clamped.(electogenic IV relationship). The reversal potential was measured when the Na gradient was varied.

Question 124

Does the N terminus account for the stoichiometry difference of NBC? Results:

Answer 124

PCT cells: Vrev= 20mv (when Nai=0 and Nao=50) Stoichiometry =1Na: 3HCO. CD cells: Vrev= 40mv = 1Na: 2 HCO. Therefore, its not the N terminus that determines the change in stoichiometry, but where the channels are expressed.

Question 125

What does cause the stoichiometry difference in the NBC?

Answer 125

Tested Protein kinase phosphorylation. Before 1:3, after 1:2. In a mutant with an inactive PKA site, the channel was stuck at the 1:3 stoichiometry. In a mutant that mimics phosphorylation, stuck in the 1:2 stoichiometry. PKA stimulates HCO3- secretion.

Question 126

NBC stoichiometry changes with phosphorylation, but also what? Know how?

Answer 126

Calcium levels. In xenopus experiments: - low Ca- Vrev -70, 1:2. -High Ca- Vrev -40, 1:3. Calcium stimulates HCO-3 reabsorption.